γ-Enolase, a glycolytic enzyme, is expressed specifically in neurons. It exerts neurotrophic activity and has been suggested to regulate growth, differentiation, survival and regeneration of neurons. In the present study, we investigated the involvement of γ-enolase in PI3K (phosphoinositide 3-kinase)/Akt and MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) signalling, the two pathways triggered predominantly by neurotrophic factors. Whereas the PI3K/Akt pathway, rather than the MAPK/ERK pathway, is involved in γ-enolase-enhanced cell survival, γ-enolase-stimulated neurite outgrowth requires both pathways, i.e. the activation of both PI3K and ERK1/2, leading to subsequent expression of the growth-cone-specific protein GAP-43 (growth-associated protein of 43 kDa). MEK (MAPK/ERK kinase) and PI3K inhibition blocked or attenuated the neurite outgrowth associated with dynamic remodelling of the actin-based cytoskeleton. We show that γ-enolase-mediated PI3K activation regulates RhoA kinase, a key regulator of actin cytoskeleton organization. Moreover, the inhibition of RhoA downstream effector ROCK (Rho-associated kinase) results in enhanced γ-enolase-induced neurite outgrowth, accompanied by actin polymerization and its redistribution to growth cones. Our results show that γ-enolase controls neuronal survival, differentiation and neurite regeneration by activating the PI3K/Akt and MAPK/ERK signalling pathways, resulting in downstream regulation of the molecular and cellular processes of cytoskeleton reorganization and cell remodelling, activation of transcriptional factors and regulation of the cell cycle.